Printer Friendly

Budlein a from Viguiera robusta inhibits leukocyte-endothelial cell interactions, adhesion molecule expression and inflammatory mediators release.


Budlein A has been reported to exert some analgesic and anti-inflammatory properties. In this study, we have evaluated its effect on LPS-induced leukocyte recruitment in vivo and the mechanisms involved in its anti-inflammatory activity. In vivo, intravital videomicroscopy was used to determine the effects of budlein A on LPS-induced leukocyte-endothelial cell interactions in the murine cremasteric microcirculation. In vitro, the effects of budlein A on LPS-induced cytokine, chemokine and nitrites release, T-cell proliferative response as well as cell adhesion molecule expression (CAM) were evaluated. In vivo, intraperitoneal administration of budlein A (2.6 mM/kg) caused a significant reduction of LPS-induced leukocyte rolling flux, adhesion and emigration by 84, 92 and 96% respectively. In vitro, T-cell proliferative response was also affected by budlein A. When murine J774 macrophages were incubated with the sesquiterpene lactone, LPS-induced IL-1[beta], tumor necrosis factor-[alpha] (TNF-[alpha]) and keratinocyte-derived chemokine (KC) release were concentration-dependently inhibited. In human umbilical vein endothelial cells (HUVECs), budlein A also reduced the production of TNF-[alpha], monocyte chemoattractant protein-1 (MCP-1), IL-8, nitrites and CAM expression elicited by LPS. Budlein A is a potent inhibitor of LPS-induced leukocyte accumulation in vivo. This effect appears to be mediated through inhibition of cytokine and chemokine release and down-regulation of CAM expression. Thus, it has potential therapeutic interest for the control of leukocyte recruitment that occurs in different inflammatory disorders.

[c] 2009 Elsevier GmbH. All rights reserved.

Keywords: Budlein A; Viguiera robusta; Leukocyte-endothelium; LPS-stimulated cells; Cytokines; Chemokines


Inflammatory response is associated to several diseases such as sepsis, atherosclerosis, allergy, autoimmunity, graft rejection, and cancer (Abou-Raya and Abou-Raya 2006; Clavijo-Alvarez et al. 2007). To date there is not an effective therapy for the control of the inflammatory process due to the peculiarity of the immune system mediators and the cell subtype involved in each inflammatory disease (Henson 2005). Therefore, there is a clear need in discovering more efficient and selective anti-inflammatory drugs that display fewer side effects than glucocorticoids. The rational studies that search new potential anti-inflammatory drugs are based on the discovery of novel biochemical targets (Krakauer 2004) and genomic approaches (Calvano et al. 2005). However, relevant compounds found in traditional herbs or new plants (Nam 2006; Yuan et al. 2006) can constitute an alternative source of anti-inflammatory drugs and the research in this area is of increased interest in the recent years.

Sesquiterpene lactones are secondary metabolites commonly found in plants from the Asteraceae family that can display a broad array of biological/therapeutical activities (Pickman 1984; Da Costa et al. 1998; Villarreal et al. 1994). Budlein A is a sesquiterpene lactone of the furanoheliangolide subtype (Fig. 1) (De Vivar et al. 1976). It was isolated from different Viguiera species (De Vivar et al. 1976; Valerio et al. 2007). Previous studies have shown that budlein A can exert antibacterial and antitumor activity (Arakawa et al. 2003; Da Costa et al. 1998), and a recent study has shown in mice its antinociceptive and anti-inflammatory activity (Valerio et al. 2007).


At sites of acute inflammation, circulating neutrophils emigrate from the blood stream into the extravascular tissues through the postcapillary venules. The migration of leukocytes from the blood to sites of extravascular injury is mediated through a sequential cascade of leukocyte-endothelial cell adhesive interactions which involve an array of cell adhesion molecules (CAMs) present on leukocytes and endothelial cells. This multi-step process is initiated by the tethering of leukocytes to the endothelium, followed by weak, transient adhesive interactions manifested as leukocyte rolling, leading ultimately to firm leukocyte adhesion to and subsequent transmigration through the vascular endothelium (Springer 1994; Ley et al. 2007). In addition to CAMs, chemoattractant molecules such as chemotactic cytokines or chemokines, have the potential to recruit specific cell types and are involved in the regulation of leukocyte trafficking (Baggiolini 1998). Chemokines are small, structurally related, disulfide-linked polypeptides that are potent mediators of cell adhesion and migration through their interactions with a family of G-protein-coupled receptors expressed on leukocytes. Several CXC chemokines such as interleukin-8 (IL-8; CXCL8) preferentially recruit neutrophils whereas CC, C and [CX.sub.3]C chemokines preferentially attract lymphocytes and monocytes (Baggiolini 1998; Laudanna et al. 2002).

Therefore, in the present study we have extended previous findings and evaluated the effect of budlein A on LPS-induced leukocyte-endothelial cell interactions within the murine cremasteric microcirculation and its possible immunosuppressive effect on mouse splenic cells. To extend these findings to humans, we have also assessed the effect of this sesquiterpene lactone on cytokine/chemokine and NO release as well as on CAM expression induced by LPS-stimulated human endothelial cells.

Materials and methods

Budlein A isolation

Budlein A was isolated from the leaves of Viguiera robusta Gardn., as previously described (Valerio et al. 2007). Its chemical structure was determined by means of spectrometric analysis, i.e. IR and [sup.1.H] and [sup.13.C] nuclear magnetic resonance (NMR) spectrometry as well as comparison with an authentic sample and data reported in the literature (MW=374.1365) (Da Costa et al. 2001). The analytical procedures indicated that the purity of the budlein A used in this study was between 95-98%.


BALB/c and C57BL/6 mice were from Jackson Laboratories (Bar Harbor, ME) and from Charles River Laboratories (L'Arbresle, France). The animal colony were bred and maintained under specific pathogen-free conditions. For all the experimental period the mice were fed with autoclaved balanced diet and water. All the protocols used in this study were approved by the Ethics Committee of Animal Experimentation (05.1.483.53.9), Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, University of Sao Paulo, Brazil and Animal Care and Use Committee, Faculty of Medicine, University of Valencia, Spain. The animals used were 22-30 g weight.

Intravital microscopy

The mouse cremaster preparation used in this study was similar to that described previously (Sanz et al. 2001). C57BL/6J mice were anesthetized by i.p. injection with a mixture of xylazine hydrochloride (l0mg/kg) and ketamine hydrochloride (200mg/kg). A polyethylene catheter was placed in the jugular vein to permit the intravenous administration of additional anesthetic. The cremaster muscle was dissected free of tissues and exteriorized onto an optical clear viewing pedestal. The muscle was cut longitudinally with a cautery and held fiat against the pedestal by attaching silk sutures to the corners of the tissue. The muscle was then perfused continuously at a rate of 1 ml/min with warmed bicarbonate-buffered saline (pH 7.4). The cremasteric microcirculation was then observed by using an intravital microscope (Nikon Optiphot-2, SMZ1, Bad-hoevedorp, Netherlands) equipped with a 50x objective lens (Nikon SLDW, Badhoevedorp, Netherlands) and a l0x eyepiece. A video camera (Sony SSC-C350P, Koeln, Germany) mounted on the microscope projected the image onto a color monitor and the images were video recorded for playback analysis. Single unbranched cremasteric venules (20-40 [micro]m in diameter) were selected for study and the diameter was measured on-line by using a video caliper (Microcirculation Research Institute, Texas A&M University, College Station, Texas). Centerline red blood cell velocity ([V.sub.rbc]) was also measured on-line by using an optical Doppler veloci-meter (Microcirculation Research Institute, Texas A&M University, College Station, Texas). Venular blood flow was calculated from the product of mean red blood cell velocity ([V.sub.mean] = [V.sub.rbc]/1.6) and cross sectional area, assuming cylindrical geometry. Venular wall shear rate ([gamma]) was calculated based on the Newtonian definition:[gamma]= 8 x ([V.sub.mean]/Dv) [s.sup.-1], in which [D.sub.v] is venular diameter (House and Lipowsky 1987).

The number of rolling, adherent and emigrated leukocytes was determined off-line during playback of videotaped images. Rolling leukocytes were defined as those white blood cells moving at a velocity less than that of erythrocytes in the same vessel. Leukocyte rolling velocity ([V.sub.wbc]) was determined from the time required for a leukocyte to move along 100 urn length of the microvessel and is expressed as [mu]m/s. Flux of rolling leukocytes was measured as those cells that could be seen moving past a defined reference point in the vessel. The same reference point was used throughout the experiment because leukocytes may roll for only a section of the vessel before rejoining the blood flow or becoming firmly adherent. A leukocyte was defined as adherent to venular endothelium if it was stationary for at least 30 s. Leukocyte adhesion was expressed as the number per 100 [micro]m length of venule. Leukocyte emigration was expressed as the number of white blood cells per microscopic field surrounding the venule.

Experimental protocol

Anesthetized animals were injected locally by s.c. injection beneath the scrotal skin using a 30-gauge needle with 0.1ml (each testicle) of sterile saline or LPS solution. Preliminary experiments indicated that local administration of 0.05 [micro]g/kg LPS was optimal for examination of leukocyte-endothelial interaction (Andonegui et al. 2002). Animals were returned to their cages for 3.5 h and the right cremaster muscle was then prepared for intravital microscopy. After 4h of the intraescrotal injection of the agent under investigation, measurements of leukocyte rolling flux, velocity, adhesion, emigration, [V.sub.rbc], shear rate and diameter were obtained and recorded for 5 min. These animals received an i.p. injection of 0.3 ml of a dimethyl sulfoxide (DMSO) solution (2% in saline) 30 min prior stimulus administration. In another set of experiments, budlein A (lmg/kg or 2.6mM/kg) dissolved in DMSO (2% in saline) was administered i.p. 30 minutes before the intraescrotal injection of LPS and responses were evaluated 4h later.

Cytotoxicity assay

The cytotoxic activity was determined in J774 macrophages obtained from European Collection of Animal Cell Cultures (Salisbury, UK) and assessed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Briefly, cells were cultured in appropriate flasks and maintained in continuous exponential growth. Then, they were removed from the flasks, washed and placed in 96-well plates at a density of 1 x [10.sup.5] cells/well. The doses of budlein A (2.6-260 [micro]M) were dissolved in DMSO (0.02-2% diluted in the culture medium). Macrophages were cultured with or without different concentrations (10-1,000 [micro]M) of paclitaxel (PX) (Zodiac Prods. Farms., Sao Paulo, Brazil), used as positive control of cytotoxicity, and incubated for 24 h. At the end of the treatment period, 10 [m]l of MTT (5mg/ml) was added to the wells and cells were incubated for a further 4h. Finally, 50 [micro]l of 20% sodium dodecyl sulfate solution (SDS) was added to each well. Formazan crystals were dissolved at 37 [degrees]C, overnight. The absorbance of each well was read on a microplate reader ([mu]QUANT, Biotek Instruments Inc.) at 570 nm. Cytotoxicity rate was calculated as follows: % compound cytotoxicity = 1- (mean) O.D. drug treated/ (mean) O.D. of cells cultured in medium +2% DMSO x 100.

Proliferation assay

Spleen cells were obtained as previously described (Cardillo et al. 1998). Briefly, mice were killed by [CO.sub.2] instillation, spleens were collected and maintained in iced buffered saline solution. 4 x [10.sup.5] splenocytes were cultured in quintuplicate in flat 96-microwell plates with supplemented RPMI medium. Cells were cultured for In with medium with 2% DMSO, 0.26-26[micro]M (or 0.1-10 [micro]g/ml) budlein A dissolved in DMSO (0.02-2% in the culture medium) or 10[micro]M Dexamethasone at 37 [degrees]C. Then the cells were transferred to wells previously coated or not with 2 [micro]g/ml anti-CD3[pounds sterling] mAb. After 44 h, 10 [micro]l MTT (5mg/ml) were added to each well and cells were incubated for an additional 4h. After this period of time, 50 [micro]l of 20% SDS in PBS were added to each well and kept in the dark overnight. Absorbance was measured at 570 nm using an automatic microplate reader ([mu]QUANT, Biotek Instruments, Inc.). Results were expressed as stimulatory index (SI) = O.D of untreated or treated cells stimulated with an anti-CD3 mAb/O.D. of cells cultured in medium +2% DMSO.

Measurement of cytokine and chemokine release from a murine macrophage cell line

J774 macrophages were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS, l00U/ml penicillin, l00[micro]g/ml streptomycin, 2mM L-glutamine, 1mM HEPES and 5mM sodium pyruvate at 37 [degrees]C in a 5% [CO.sub.2] humidified atmosphere. Cultured cells were removed from flasks and washed twice with PBS. Cell pellets were suspended in supplemented DMEM and counted in an hemocytometer. Viability was evaluated by trypan blue exclusion.

Cells (1 x [10.sup.6] cells/well) were cultured in 24-well plate (Nalge Nunc, Rochester, NY) in 1 ml of supplemented DMEM and allowed to adhere for 1 h. The medium was removed and replaced with fresh medium. The cells were then incubated for another 1 h in medium with 2% DMSO, 0.26-26 [micro]M (or 0.1-10 [micro]g/ml) budlein A dissolved in DMSO (0.02-2% in the culture medium) or with 10 [micro]M Dexamethasone. Thereafter, the cells were stimulated with 1 [micro]g/ml LPS for 24 h at 37 [degrees]C. At the end of the experiment, cell-free super-natants were collected for IL-1[beta], TNF-[alpha and KC ELISA assays.

Measurement of cytokine and chemokine release from human umbilical vein endothelial cells (HUVECs) stimulated with LPS

HUVECs were isolated by collagenase treatment (Jaffe et al. 1973) and maintained in human endothelial cells specific medium EBM-2 supplemented with EGM-2 and 10% FCS. Cells up to passage 2 were grown to confluence in 24-well culture plates. Prior to every experiment, cells were incubated for 16 h in medium containing 1% FCS and then returned to the 10% FCS medium at the beginning of all experimental incubations. HUVECs were seeded in 24-well plate and stimulated with LPS 1 [micro]g/ml for 4h. Vehicle (2% DMSO), Budlein A (0.26-26 [micro]M or 0.1-10[micro]g/ml) dissolved in DMSO (0.02-2% in culture medium) or Dexamethasone (10 [micro]M) were added to some wells 1 or 2h prior to LPS stimulation. At the end of the experiment, cell-free supernatants were collected for cytokine and chemokine conventional sandwich ELISA assays.

Nitric oxide production by HUVECs

Nitric oxide (NO) production by LPS-stimulated HUVECs was measured by the Griess reaction. The protocol followed was identical to that described above. Cell free supernatants (0.1 ml) were incubated with equal volumes of Griess reagent mixtures (1% sulfanilamine, 0.1% N- (l-naphtyl)-ethylendiamine dihydrochloride, 2.5% [H.sub.3.PO.sub.4]) at room temperature for l0min. The absorbance was measured in a microplate reader at 540 nm. NO concentrations were calculated from a sodium nitrite standard curve. Data were presented as [mu]M concentration of [] (nitrite).

Immunofluorescence assay

E-selectin, ICAM-1 and VCAM-1 were visualized in HUVEC monolayers by immunofluorescence. HUVECs were stimulated with LPS 1 [micro]g/ml for 4h. Vehicle (2% DMSO) or Budlein A (2.6 [micro]M dissolved in 0.2% DMSO) were added to some wells 1 h prior to LPS stimulation. At the end of the experiment, cells were fixed with 4% paraformaldehyde. Cells were blocked with glycine 0.1% and then incubated for 1 h with a monoclonal antibody against either E-selectin (dilution 1/250), ICAM-1 (dilution 1/250) or VCAM-1 (dilution 1/250), followed by incubation with a rabbit anti-mouse FITC-conjugated secondary antibody (dilution 1/1500) at room temperature for lh in the dark. DAPI staining was applied to visualize cells nuclei.

Determination of synergistic effect between Budlein A and Dexamethasone

In order to investigate whether a synergistic effect could be found using Budlein A in combination with small concentration of Dexamethasone, we conducted an in vivo cell recruitment assay applying the Berenbaum's isobol method, as described (Wagner and Ulrich-Merzenich 2009). Briefly, C57BL/6 mice (5 per group) were euthanized by [CO.Sub.2] instillation and peritoneal lavage fluid was collected from the control (saline injected) and 1 h pretreated animals (0.3 ml, i.p.) with Budlein A (2.6mM/kg, dissolved in saline), Dexamethasone (1 mg/kg, dissolved in saline) or a combination dose (0.26 mM/kg of Budlein A + 0.01 mg/kg of Dexamethasone). Then, mice were i.p. LPS-stimulated (0.3 ml, 0.05([micro]g/kg). Four hours later, cells from the peritoneal cavity were harvested by injection of 3 ml of saline added with heparin (5U/ml). The abdomens were gently massaged and a blood-free cell suspension was carefully withdrawn with a syringe. Total cells (leukocytes) counts were immediately performed in a Neubauer chamber. A x400 magnification under optical microscopy was used.


HEPES, sodium pyruvate, LPS of E. coli (serotype 0127:B8), BSS, Dexamethasone, MTT and Griess reagent mixtures were from Sigma Chemical Co., St. Louis, MO. Penicillin, streptomycin and L-glutamine were from Gibco-Invitrogen, Carlsbad, CA. Mouse IL-l[beta], TNF-[alpha], KC, their antibodies pairs and the anti-CD3[member.of] mAb were from BD Pharmingen, San Diego, CA. EBM-2 medium supplemented with EGM-2 was from Clonetics, Barcelona, Spain. Ficoll Hypaque density gradient was from GE Healthcare-Pharmacia, Uppsala, Sweden. Human TNF[alpha], IL-8 and MCP-1 were from PeproTech, London, UK. The antibody pairs for human TNF[alpha], IL-8 and MCP-1 ELISA were from R&D Systems, Madrid. Neutravidin-horseradish peroxidase was from Perbio Science, Cheshire, UK. K-Blue substrate was from Neogen, Lexington, KY. Anti-human E-selectin (1.2B6, mouse Igd), ICAM-1 (6.52B5, mouse [IgG sub 1]) and VCAM-1 (1.G11B1, mouse [IgG.sub.1]) were from Serotec, Madrid, Spain. The rabbit anti-mouse FITC-conjugated secondary antibody was from DakoCytomation, Glostrup, Denmark.

Statistical analyses

All data are expressed as mean [+ or -]SEM. The data within groups were compared using a one-way analysis of variance (ANOVA) with a Newman-Keuls post hoc correction for multiple comparisons. In some experiments the data within groups were compared using a one-way ANOVA analysis applying Tukey and Bonferroni corrections. A p value <0.05 was considered statistically significant.


Effect of the budlein A pretreatment on leukocyte-endothelial cell interactions

Intravital microscopy was used to examine Ieukocyte-endothelial cell interactions in the cremasteric microcirculation. Fig. 2 shows the effect of budlein A on subacute LPS-induced leukocyte responses. After 4h intraescrotal injection of 0.1 ml of 0.05[micro]g/kg LPS, significant increases in leukocyte rolling flux (91.0[+ or -]7.8 vs. 46.0[+ or -]1.7 cells/min), adhesion (14.0[+ or -]2.8 vs. 1.0[+ or -]1.4 cells per 100 urn vessel) and emigration (20.5[+ or -] 0.7 vs. [1.3[+ or -]1.0 cells per field) and significant decreases in the leukocyte rolling velocity (8.4[+ or -]1.9 vs. 25.9[+ or -]3.3[micro]m/s) were detected vs. values obtained in the saline treated control group. Budlein A pretreatment at 1 mg/kg (or 2.6mM/kg, 0.3 ml) significantly reduced LPS-induced leukocyte rolling flux, adhesion and emigration by 84, 92 and 96% respectively after 4 h exposure to LPS (Fig. 2) and significantly increased the reduction in the leukocyte rolling velocity. None of these treatments had significant effects on shear rate (Table 1). The i.p. administration of vehicle (2% DMSO) did not exert any significant effect on the responses elicited by the intraescrotal administration of saline or LPS.

Table 1. Hemodynamic parameters in the mice cremasteric

               Saline               LPS                LPS+budlein A

Venular         26.5[+ or -]4.7    28[+ or -]3.4       29[+ or -]5.3

Venular shear  618.7[+ or -]6.2  784[+ or -]11.3  733.6[+ or -]230.5
rate ([s sup

Parameters (mean+ or -]SEM, in animals used for intravital microscopy
studies) were measured 4 h after the intraescrotal
injection of saline, LPS (0.05 [micro]g/kg) or LPS + budlein A
(2.6 mM/kg); (n=4), from two different experiments.

Cytotoxic activity of budlein A

To assess the potential toxic effects of budlein A, a cytotoxicity assay on J774 macrophage cell line using the doses employed in our studies was carried out. Budlein A at 2.6 and 26[micro]M was not able to induce any toxic effect on these cells after 24 h incubation (Fig. 3). Only the highest dose assayed (260.[micro]M) displayed a mild cytotoxic effect (17.6%) when compared to paclitaxel at 100 and 1,000 [micro]M (68.8 and 74%, respectively). Paclitaxel was used as a positive control on this assay.


Budlein A inhibits inflammatory mediators release from murine macrophages

In addition to cell adhesion molecules, chemoattractant molecules such as chemokines are involved in the regulation of leukocyte trafficking. Therefore, we next investigated whether budlein A can modulate the release of inflammatory mediators such as cytokines and chemokines from murine macrophages. As it is shown in Fig. 4, LPS-stimulated J774 macrophages secreted high levels of IL-l[beta]TNF-[alpha] and KC Budlein A inhibited the release of these mediators induced by LPS in a concentration-dependent manner (Fig. 4). As expected, pre-treatment of the cells with Dexamethasone also significantly reduced the generation of IL-l[beta], TNF-[alpha] and KC elicited by LPS. LPS-induced responses were not affected by the incubation with 2% DMSO.


Inhibitory activity of budlein A on T cell proliferation

In order to elucidate the possible effect of budlein A on adaptive immune response, we isolated murine splenic T lymphocytes. Budlein A displayed a strong inhibitory activity on T cell proliferation induced by anti-CD3 stimulation. Indeed, the sesquiterpene lactone inhibited this response in a concentration-dependent manner (Fig. 5). In contrast, Dexamethasone at 10 [micro]M only partially inhibited T-cell proliferation induced by anti-CD3. Incubation with 2% DMSO did not affect T cell proliferation.


Budlein A decreases the levels of cytokines/chemokines and nitrites released from HUVECs stimulated with LPS

To extend these findings to human, we measured the production of TNF-[alpha], IL-8, MCP-1 and nitrites in response to LPS in culture of HUVECs at 4h. There was a significant release of these inflammatory mediators after 4h stimulation with 1 [micro]g/ml of LPS (Fig. 6). Budlein A inhibited in a concentration-dependent manner the release of TNF-[alpha], MCP-1 and nitrites (Fig. 6A, C and D), whereas LPS-induced IL-8 production was only inhibited by budlein A at the highest dose assayed (26 [micro]M). Preincubation for 2h with l0 [micro]M Dexamethasone did not significantly affect the synthesis and release of TNF-[alpha] but it did the synthesis of IL-8, MCP-1 and nitrite elicited by LPS. In these assays, the incubation with 2% DMSO did not affect LPS-induced mediators release.


Budlein A reduces LPS-induced adhesion molecules expression from HUVECs

As it is illustrated in Fig. 7, stimulation of HUVECs with LPS for 4h resulted in the increased expression of E-selectin, ICAM-1 and VCAM-1. Pretreatment of the cells with 2.6 [micro]M budlein A markedly reduced LPS-induced ICAM-1 and VCAM-1 expression while E-selectin expression was not affected (Fig. 7).


Budlein A and Dexamethasone inhibit leukocytes recruitment in an antagonistic manner

In order to evaluate a possible synergistic effect between Budlein A and Dexamethasone, a simple determination was made based on the in vivo cell recruitment assay. Mice that received saline and were i.p. LPS-stimulated (control) presented the highest number of leukocytes recruited to peritoneal cavity. All pretreated mice (Budlein A and Dexamethasone alone or in combination) had the number of recruited cells diminished (Fig. 8). When we observed the effect of the compounds on the inhibition of leukocytes recruitment (compared to LPS group), an antagonistic effect between Budlein A and Dexamethasone could be detected applying the Berenbaum's method. The inhibition of cell recruitment under the effect of a combination dose was lower than Budlein A and Dexamethasone doses used alone (3.18 vs. 5.32 x [10.sup,6] leukocytes/ml).



In the present study, we have demonstrated that the sesquiterpene lactone budlein A displays anti-inflammatory activity through the inhibition of LPS-induced leukocyte-endothelial cell interactions in vivo and CAM expression as well as cytokine and chemokine release in vitro. Additionally, it also displays an anti-proliferative response on T-cells. Interestingly, budlein A treatment inhibited the leukocyte rolling flux to levels lower than the group treated with saline solution (Fig. 2A). This fact can be explained by the existence of a basal P-selectin expression in the cremasteric microcirculation (Hickey et al. 1999). Therefore, it is tempting to speculate that budlein A maybe decreases basal rolling flux by reducing the expression of P-selectin.

Vascular endothelium is the main controller of leukocyte traffic between the bloodstream and the extravascular space. In fact, the migration of leukocytes from the blood to sites of extravascular injury is mediated through a sequential cascade of leukocyte-endothelial cell adhesive interactions, which involve an array of cell adhesion molecules present on leukocytes and endothelial cells (Cook-Mills and Deem 2005). In this regard, we could get conclusive data showing that budlein A is partly able to reduce ICAM-1 and VCAM-1 expression when human endothelial cells were stimulated with LPS (Fig. 7). Although this finding is relevant, it might be possible that budlein A can control the inflammatory process by inhibiting the release of other mediators involved in leukocyte trafficking such as cytokines and chemokines.

A large number of studies have demonstrated that LPS increases microvascular permeability, neutrophil chemotaxis, CAM expression, the release of proinflammatory cytokines such as TNF-[alpha], IL-l[beta] chemokines and NO (Andonegui et al. 2002; Calvano et al. 2005). In this context, in our study budlein A inhibited in a concentration-dependent manner the IL-l[beta], TNF[alpha] and KC release by LPS-stimulated J774 macrophages. In human cells, we also evaluated the effect of budlein A on LPS-induced production of different cytokines, chemokines and nitrites using endothelial cells. Budlein A inhibited in a concentration-dependent manner LPS-induced TNF-[alpha], MCP-1 and nitrites release from HUVECs.

Since some of the previous findings might occur as a consequence of a potential cytotoxic effect exerted by budlein A and previous studies have shown that high doses of other sesquiterpene lactones can display cytotoxic/apoptotic effects on PBMC or splenic cells (Cho et al. 2000; Dirsch et al. 2001), we next investigated such possibility. We first observed that at the doses employed in the present study budlein A did not cause considerable toxic effect on murine macrophages. In fact, it was necessary to use a dose 10 fold higher (260 [micro]M) than the highest dose used in our assays to detect a mild cytotoxic effect (Fig. 3). Furthermore, budlein A cytotoxicity was neither detected in other studies (Huacuja et al. 1993) nor for the concentrations used in our experimental protocols (0.26-26 [micro]M).

The anti-inflammatory activity of budlein A found in this study could be due to nuclear factor-kB (NF-kB) inhibition. In this context, several sesquiterpene lactones, including budlein A, are capable to inhibit this transcription factor by the alkylation of the cystein residue in the p65 subunit (Lyss et al. 1998; Siedle et al. 2004). In fact, these natural compounds can inhibit ICAM-1 expression in human lung adenocarcinoma cell line (A549), COX-2 and inducible nitric oxide synthase expression in RAW 264.7 macrophages, NO release by LPS-activated murine peritoneal macrophages and IL-1[beta] IL-2, IL-6 and TNF-[alpha] release by PBMC (Calvano et al. 2005; Hilmi et al. 2003; Yuuya et al. 1999), being most of these responses mediated through NF-kB activation.

On the other hand, sesquiterpene lactones such as parthenolides have been reported to inactivate mitogen activated protein kinases (MAPKs) by irreversibly binding to the thiol (-SH) groups (Lyss et al. 1998). In this context, other studies have demonstrated that even budlein A is able to inactivate thiol groups on sperm cells without affecting cell viability (Huacuja et al. 1993). Interestingly, the results obtained in the present study also suggest that budlein A and glucocorticoids exert anti-inflammatory activity through different mechanisms. Although Dexamethasone is a classical NF-kB inhibitor (Tuckermann et al. 2005), it requires several hours to elicit its anti-inflammatory effects. In contrast, it seems that budlein A can more rapidly inactivate different inflammatory cascades since 1 h pre-incubation was enough to inhibit LPS-induced cytokine/chemokine synthesis and release. Moreover, no synergistic effect between Budlein A and Dexamethasone was observed when an in vivo cell recruitment assay was conducted (Fig. 8). It seems that these compounds present their anti-inflammatory effects independently. Indeed, sesquiterpene lactones can bind to NF--kB and inhibit I--kB kinase (Lyss et al. 1998). Therefore, it might be possible that budlein A does not share with glucocorticosteroids their undesirable side effects.

In conclusion, in the present study we have extended previous findings and demonstrate that budlein A inhibits leukocyte-endothelial cell interactions in vivo. This effect is partly mediated through the inhibition of cytokines/chemokines release, CAM expression and NO production. Furthermore, we have also provided evidence of its immunosuppressive activity in murine splenic cells. These results suggest that budlein A may have a therapeutical potential in the control of the inflammatory process associated to different acute and chronic inflammatory disorders.


The present study has been supported by grant SAF 2008-03477 from CICYT, Spanish Ministry of Education and Science; Research Group 03/166 of Conselleria of Education and Culture (Generalitat Valenciana); Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) (Brazil). RN was supported by a grant from Programa de Mobilidade Internacional, Banco Santander. CR was supported by a grant from Spanish Ministry of Education and Science. NSA was supported by a grant from FAPESP. PJJ was supported by a grant from the University of Valencia, Spain.

Conflicts of interest

No conflict to disclose.


Abou-Raya, A., Abou-Raya, S., 2006. Inflammation: a pivotal link between autoimmune diseases and atherosclerosis. Autoimmun. Rev. 5, 331-337.

Andonegui, G., Goyert, S.M., Kubes, P., 2002. Lipopolysac-charide-induced leukocyte-endothelial cell interactions: a role for CD14 versus toll-like receptor 4 within micro-vessels. J. Immunol. 169, 2111-2119.

Arakawa, N.S., Nihei, J.S., Da Costa, F.B., Nomizo, A., 2003. Evaluation of cytotoxic activity of budlein A isolated from Viguiera robusta Gardn. (Asteraceae). Abstract. Braz. J. Pharm. Sci. 39, 243.

Baggiolini, M., 1998. Chemokines and leukocyte traffic. Nature 392, 565-568.

Calvano, S.E., Xiao, W., Richards, D.R., Felciano, R.M., Baker, H.V., Cho, R.J., Chen, R.O., Brownstein, B.H., Cobb, J.P., Tsehoeke, S.K., Miller-Graziano, C., Mol-dawer, L.L., Mindrinos, M.N., Davis, R.W., Tompkins, R.G., Lowry, S.F., 2005. A network-based analysis of systemic inflammation in humans. Nature 437, 1032-1037.

Cardillo, F., Nomizo, A., Mengel, J., 1998. The role of the thymus in modulating gammadeita T cell suppressor activity during experimental Trypanosoma cruzi infection. Int. Immunol. 10, 107-116.

Cho, J.Y., Baik, K.U., Jung, J.H., Park, M.H., 2000. In vitro anti-inflammatory effects of cynaropicrin, a sesquiterpene lactone, from Saussurea lappa. Eur. J. Pharmacol. 398, 399-407.

Clavijo-Alvarez, J.A., Hamad, G.G., Taieb, A., Lee, W.P., 2007. Pharmacologic approaches to composite tissue allograft. J. Hand Surg. [Am.] 32, 104-118.

Cook-Mills, J.M., Deem, T.L., 2005. Active participation of endothelial cells in inflammation. J. Leukoc. Biol. 77, 487-495.

Da Costa, F.B., Ito. I.Y., Andre, R.F.G., Vichnewski. W., 1998. Constituents of Viguiera species with antibacterial activity. Fitoterapia 69, 86-87.

Da Costa, F.B., Schorr, K., Arakawa, N.S., Schilling, E.E., Spring, O., 2001. Infraspecific variation in the chemistry of glandular trichomes of two Brazilian Viguiera populations. J. Braz. Chem. Soc. 12, 403-407.

De Vivar, A.R., Diaz, C.G.E., Bratoeff, E.A., Jimenez, L., 1976. The germacranolides of Viguiera buddleiaeformis structures of budlein-A and -B. Phytochemistry 15, 525-529.

Dirsch, V.M., Stuppner, H., Vollmar, A.M., 2001. Cytotoxic sesquiterpene lactones mediate their death-inducing effect in leukemia T cells by triggering apoptosis. Planta Med. 67, 557-559.

Henson, P.M., 2005. Dampening inflammation. Nat. Immunol. 6, 1179-1181.

Hickey, M.J., Kanwar, S., McCafferty, D.M., Granger, D.N., Eppihimer, M.J., Kubes, P., 1999. Varying roles of E-selectin and P-selectin in different microvascular beds in response to antigen. J. Immunol. 162, 1137-1143.

Hilmi, F., Gertsch, J., Bremnei, P., Valovic, S., Heinrich, M., Sticher, O., Heilmann, J., 2003. Cytotoxic versus anti-inflammatory effects in HeLa, Jurkat T and human peripheral blood cells caused by guaianolide-type sesquiterpene lactones. Bioorg. Med. Chem. 11, 3659-3663.

House, S.D., Lipowsky, H.H., 1987. Leukocyte-endothelium adhesion: microhemodynamics in mesentery of the cat. Microvasc. Res. 34, 363-379.

Huacuja, R.L., Carranco, A., Guzman, S.A., Guerrero, C., 1993. Inactivation of SH groups with sesquiterpene lactones: effects on nuclear decondensation pattern/motility induced by heparin in human spermatozoa. Adv. Contracept. Deliv. Syst. 9, 97-106.

Jaffe, E.A.. Nachman, R.L., Becker, C.G., Minick, C.R., 1973. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J. Clin. Invest. 52, 2745-2756.

Krakauer, T., 2004. Molecular therapeutic targets in inflammation: cyclooxygenase and NF-kappaB. Curr. Drug Targets Infiamm. Allergy 3, 317-324.

Laudanna, C., Kim, J.Y., Constantin, G., Butcher, E., 2002. Rapid leukocyte integrin activation by chemokines. Immunol. Rev. 186, 37-46.

Ley, K., Laudanna, C., Cybulsky, M.I., Nourshargh, S., 2007. Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nat. Rev. Immunol. 7, 678-689.

Lyss, G., Knorre, A., Schmidt, T.J., Pahl, H.L., Merfort, I., 1998. The anti-inflammatory sesquiterpene lactone helenalin inhibits the transcription factor NF-kappaB by directly targeting p65. J. Biol. Chem. 273, 33508-33516.

Nam, N.H., 2006. Naturally occurring NF-kappaB inhibitors. Mini Rev. Med. Chem. 6, 945-951.

Pickman, A.K., 1984. Antifungal activity of sesquiterpene lactones. Biochem. Syst. Ecol. 12, 13-18.

Sanz, M.J., Hickey, M.J., Johnston, B., McCafferty, D.M., Raharjo, E., Huang, P.L., Kubes, P., 2001. Neuronal nitric oxide synthase (NOS) regulates leukocyte-endothelial cell interactions in endothelial NOS deficient mice. Br. J. Pharmacol. 134, 305-312.

Siedle, B., Garcia-Pineres, A.J., Murillo, R., Schulte-Monting, J., Castro, V., Rungeler, P., Klaas, C.A., Da Costa, F.B., Kisiel, W., Merfort, I., 2004. Quantitative structure-activity relationship of sesquiterpene lactones as inhibitors of the transcription factor NF-kappaB. J. Med. Chem, 47, 6042-6054.

Springer, T.A., 1994. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76,301-314.

Tuckermann, J.P., Kleiman, A., McPherson, K.G., Reichardt, H.M., 2005. Molecular mechanisms of glucocorticoids in the control of inflammation and lymphocyte apoptosis. Crit. Rev. Clin. Lab. Sci. 42, 71-104.

Valerio, D.A., Cunha, T.M., Arakawa, N.S., Lemos, H.P., Da Costa, F.B., Parada, C.A., Ferreira, S.H., Cunha, F.Q., Verri Jr., W.A., 2007. Anti-inflammatory and analgesic effects of the sesquiterpene lactone budlein A in mice: inhibition of cytokine production-dependent mechanism. Eur. J. Pharmacol. 562, 155-163.

Villarreal, M.L., Alvarez, L., Aionso, D., Navarro, V., Garcia, P., Delgado, G., 1994. Cytotoxic and antimicrobial screening of selected terpenoids from Asteraceae species. J. Ethnopharmacol. 42, 25-29.

Wagner, H., Ulrich-Merzenich, G., 2009. Synergy research: approaching a new generation of phytopharmaceuticals. Phytomedicine 16, 97-110.

Yuan, G., Wahlqvist, MX., He, G., Yang, M., Li, D., 2006. Natural products and anti-inflammatory activity. Asia Pac. J. Clin. Nutr. 15, 143-152.

Yuuya, S., Hagiwara, H., Suzuki, T., Ando, M., Yamada, A., Suda, K., Kataoka, T., Nagai, K., 1999. Guaianolides as immunomodulators. Synthesis and biological activities of dehydrocostus lactone, mokko lactone, eremanthin, and their derivatives. J. Nat. Prod. 62, 22-30.

Roberto Nicolete (a), Nilton S. Arakawa (b), Cristina Rius (c), Auro Nomizo (a), Peter J. Jose (c), Fernando B. Da Costab (b), Maria-Jesus Sanz (c), (d), Lucia H. Faccioli (a),*

(a) Departamento de Analises Clinicas, Toxicologicas e Bromatologicas, Faculdade de. Ciencias Farmaceuticas de Ribeirao Preto, Universidade de Sao Paulo, Av. do Cafe sln, 14040-903 Ribeirao Preto, Brazil

(b) Departamento de Ciencias Farmaceuticas, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Universidade de Sao Paulo, Av. do Cafe sln, 14040-903 Ribeirao Preto, Brazil

(c) Department of Pharmacology, Faculty of Medicine, University of Valencia, Avda. Blasco Ibanez 15, 46010 Valencia, Spain

(d) Ciber CB0610610027 "Respiratory Diseases" Carlos III Health Institute, Spanish Ministry of Health, Madrid, Spain

* Corresponding author. Tel: +55 16 36024303; fax: +55 16 36024725.

E-mail address: (L.H. Faccioli).

0944-7113/$ - see front matter [c] 2009 Elsevier GmbH. All rights reserved.

doi: 10.1016/j.phymed. 2009.04.002
COPYRIGHT 2009 Urban & Fischer Verlag
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2009 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Author:Nicolete, Roberto; Arakawa, Nilton S.; Rius, Cristina; Nomizo, Auro; Jose, Peter J.; Costa, Fernando
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:4EUSP
Date:Oct 1, 2009
Previous Article:Effect of Bacopa monniera on liver and kidney toxicity in chronic use of opioids.
Next Article:Curcumin inhibits cell proliferation of MDA-MB-231 and BT-483 breast cancer cells mediated by down-regulation of NF[kappa]B, cyclinD and MMP-1...

Terms of use | Copyright © 2017 Farlex, Inc. | Feedback | For webmasters